Field of the Invention
The present invention relates to the field of prenatal diagnosis of fetal aneuploidy.
In particular, the invention relates to a method for rapid prenatal diagnosis and detection of fetal aneuploidy by using a microfluidic digital PCR (polymerase chain reaction) that enables rapid, allele independent molecular detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue.
Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual compositions, components, or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Fetal aneuploidy is a chromosomal abnormality that is a common cause of genetic disorder. It is represented by an abnormal number of chromosomes, such as an extra or missing chromosome. The incidence of fetal aneuploidy and other chromosome abnormalities is approximately 9 per 1000 live births [1]. It is difficult to estimate their true incidence among all pregnancies due to the strong association with fetal miscarriage and stillbirth. The prevalence of chromosomal abnormalities clinically recognized early pregnancy loss is greater than 50%, and fetuses with aneuploidy account for 6-11% of all stillbirths and neonatal deaths [2]. Aneuploidy rates increase with advancing maternal age, yet despite advances in non-invasive prenatal screening, diagnosis of fetal chromosomal abnormalities is the most common indication for invasive prenatal testing [2].
Conventional cytogenetics is currently the gold standard for determining fetal karyotype and thereby detecting fetal aneuploidy. In this procedure, fetal cells obtained from amniotic fluid or chorionic villi are cultured and the karyotype is analyzed microscopically by observing condensed chromosomes during metaphase stage. While conventional cytogenetics can provide accurate information regarding chromosomal aberrations, it requires approximately 1-2 weeks for patients to obtain results. This time delay may result in both increased anxiety for expectant parents, and greater maternal morbidity should pregnancy termination be desired in the setting of abnormal results. Rapid and accurate molecular based detection of aneuploidy is thus highly desirable.
There have been several molecular diagnostic techniques developed for aneuploidy detection [3-5]. These techniques include Polymerase Chain Reaction (PCR), Fluorescence in Situ Hybridization (FISH), Quantitative Polymerase Chain reaction (PCR) or Short Tandem repeats, Quantitative Fluorescence PCR (QF-PCR), Quantitative real-time PCR (RT-PCR) dosage analysis, Quantitative Spectrometry of Single Nucleotide Polymorphism and Comparative genomic Hybridization (CGH). All these techniques provide tools for detection of aneuploidy, however some are invasive and most of them tend to be lengthy, labor intensive and some are allele-dependent, so that the results depend on the underlying genetics of the population.
In conventional RT-PCR, for example, one threshold cycle corresponds to a 2-fold change in copy number, making it exceedingly challenging to measure smaller changes [6], such as a 1.5-fold increase in number of a trisomic chromosome as compared to a normal disomic chromosome.
Thus it would be advantageous to have a more rapid and accurate method for detection of presence of abnormal chromosomes.
Currently, a number of rapid molecular diagnostic tests for fetal aneuploidy are available. The most widely validated ones are fluorescent in situ hybridization (FISH) [23-25], quantitative-fluorescent PCR (QF-PCR) [26-33], and multiplex ligation probe amplification (MLPA) [34-38].
In recent years, array comparative genomic hybridization (CGH) has also been introduced for the rapid prenatal diagnosis of aneuploidy and diseases associated with copy number variation [39-44]. While array CGH is able to provide genome-wide information on copy number variations at relatively high resolution, it requires several days for analysis and substantial amount of genetic materials [39, 44].
One of the methods that has been developed recently is a digital PCR. In digital PCR the amount of nucleic acids is quantified by counting amplification from single molecules [7, 8].
Digital PCR was first used on a multi-well plate format to detect mutations and allelic imbalances associated with cancer development [13-15], and this format has recently been applied to measure allelic imbalance in placental RNA with the goal of developing a noninvasive diagnostic for trisomy 21 [16]. A microemulsion platform was developed to increase the scale of the assay [17, 18], and it is now being used as a sample preparation technique for massively parallel sequencing [19]. However, all these previously described methods are cumbersome to implement, take a long time and require significant labor.
The emergence of microfluidics has led to the development of a commercially available microfluidic digital PCR platform that enables the simultaneous performance of approximately 9000 PCR reactions [20]. It has been used to study the gene expression of single progenitor cells [12], to relate gene function to identity in environmental microbes [21], and to measure trisomy in human cell lines [22].
It is therefore an object of this invention to provide a faster and more reliable method for detection of fetal aneuploidy, which can also be combined with other molecular tests. It is shown below that using a microfluidic digital PCR assay permits diagnosis of aneuploidy in amniotic fluid and chorionic villi within six hours.
Specific Patents and Publications
Han U.S. Pat. No. 5,888,740 discloses a method for detection of fetal aneuploidy using synthetic internal controls that provide accuracy in determining chromosome copy number by strictly controlling the quantity of the internal control sequences and relative rates of polymerase chain reaction (PCR) for test versus control sequences.
Fan et al. US 2007/0202525 A1 published Aug. 30, 2007, discloses that digital PCR can be used to detect aneuploidy, such as the trisomy that causes Down Syndrome, in a mixture of fetal and maternal DNA.
Lo et al., “Digital PCR for the molecular detection of fetal chromosomal aneuploidy,” PNAS, August 7, 2007 vol. 104 no. 32 13116-13121 (Ref. [16]), discloses a digital PCR method which uses a real-time PCR assay. One method was termed “digital RCD,” or digital relative chromosome dosage. A value was calculated by dividing the number of wells positive for the chromosome 21 locus by the total number of informative wells.
Fan and Quake, “Detection of aneuploidy with digital polymerase chain reaction,” Anal Chem., 2007 Oct. 1, 79(19):7576-9, discloses that dPCR is generally applicable to any aneuploidy, does not depend on allelic distribution or gender, and is able to detect signals in mixtures of maternal and fetal DNA.
Mann et al., “Strategies for the rapid prenatal diagnosis of chromosome aneuploidy,” European Journal of Human Genetics, (2004) 12, 907-915 discloses a one-tube QF-PCR test using the relative quantification of microsatellite alleles.